This invention relates to the purification and use of a retinal pigmented epithelium derived neurotrophic factor (PEDF). This invention also relates to a truncated version of PEDF that is referred to as PEDF-BH. In addition to PEDF and PEDF-BH and functionally equivalent proteins, this invention relates to nucleic acids that encode PEDF, PEDF-BH and functionally equivalent proteins, to vectors comprising such nucleic acids, to host cells into which such vectors have been introduced, and to the use of these host cells to produce such proteins.
Many types of neurons depend upon the availability of special regulatory molecules, known as neurotrophic factors, for their survival and well-being. The best characterized of the neurotrophic factors is nerve growth factor (NGF). NGF regulates the survival and specialized function of sympathetic and dorsal root ganglion neurons in the peripheral nervous system and of some cholinergic neurons in the central nervous system. Trophic factors, which act on other neurons, have also been identified, and two such factors, ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) have been purified. Moreover, it has recently been shown that some growth factors, such as fibroblast growth factor (FGF) and epidermal growth factor (EGF), which initially were identified based on their mitogenic effects upon cells, also function as survival-promoting agents for some neurons. Post-synaptic target cells and satellite cells, such as glial cells, appear to be major sources of neurotrophic factors.
It has been proposed that the survival of retinal photoreceptor cells may also be regulated by specific neurotrophic factors. Evidence supporting this concept includes the observation that photoreceptors undergo developmental neuronal death in some species, a phenomenon which is generally considered to reflect the limited availability of neurotrophic factors. Photoreceptor development, as well as maintenance of normal function, has also been shown to require interactions with the retinal pigment epithelium (RPE), suggesting that RPE-derived molecules or factors could be necessary for photoreceptor function and survival.
The RPE develops in advance of and lies adjacent to the neural retina. A closed compartment between the two cell layers contains the interphotoreceptor matrix, and many soluble secretory products of RPE and neural retina cells are contained in the interphotoreceptor matrix. Nutrients, metabolites or trophic factors exchanged between the RPE and neural retina, must pass through the interphotoreceptor matrix. RPE cells, for example, are thought to synthesize and secrete a photoreceptor survival-promoting factor (PSPA).
Cultured RPE cells synthesize a number of well known trophic factors, including platelet derived growth factor (PDGF), FGF, transforming growth factor-xcex1 (TGF-xcex1), and transforming growth factor-xcex2 (TGF-xcex2). It is possible that these or other unknown factors derived from RPE could influence the development of the neural retina.
The neural-derived RPE forms a monolayer of cells interposed between the neural retina and circulating blood within the choroid. In this strategic location, the RPE forms a part of the blood-retina barrier, performs functions essential to retinal integrity and functions, and plays important roles in vascular, inflammatory, degenerative, and dystrophic diseases of the retina and choroid. The functions of the RPE in relation to the visual process are several-fold and include light-dark adaption, phagocytosis of shed photoreceptor outer segment membrane and nutrition. On the other hand, the close interdependence of the RPE and the neural retina during normal development has been known for a long time, but functionally is not well understood, although it is known that the RPE is important for retinal regeneration. It has been consistently observed that loss of contact of the neural retina with the RPE of many vertebrates (retinal detachment) results in degeneration of the retina. As a side effect of the retinal detachment, strong cell proliferation, originating from the RPE which underlies the areas of detachment, has often been observed.
Thus, identification of hypothetical survival-promoting factors for photoreceptor cells would potentially be of great importance for the treatment of pathological conditions which result in blindness due to photoreceptor degeneration of unknown etiology. While these types of selective photoreceptor degenerations could be due to a variety of different mechanisms, analogies with neuronal degenerations in other regions of the nervous system suggest the possible involvement of a neurotrophic activity in the retina.
The present invention relates to a purified retinal pigmented epithelium derived neurotrophic factor composition and a method for purifying such a retinal pigmented epithelium neurotrophic factor. The purification procedure comprises providing an impure protein fraction containing retinal pigmented epithelium derived neurotrophic factor and applying the impure protein fraction containing retinal pigmented epithelium derived neurotrophic factor to a cation-exchange chromatography medium. The cation-exchange chromatography medium is then washed to elute any unbound proteins and the retinal pigmented epithelium derived neurotrophic factor is eluted from the cation-exchange chromatography medium and collected.
The present invention also relates to a recombinant DNA molecule comprising a gene encoding a retinal pigmented epithelium derived neurotrophic factor having the DNA sequence or the amino acid sequence in SEQ ID NO:1 and to an organism transformed with a recombinant DNA molecule comprising a retinal pigmented epithelium derived neurotrophic factor gene having a DNA sequence identified in SEQ ID NO:1.
The present invention also relates to a method of treating tumors, ocular diseases and conditions resulting from the activity of serine proteases which comprises administering PEDF.
Cells of the retinal pigmented epithelium are closely associated with differentiating retinoblasts in vivo and contribute to an environment essential to their development and normal function, both in vivo and in vitro. Retinoblastoma cells exhibit a multi-potential differentiative nature paralleling that of their precursor, the primitive retinoblast, as evidenced by the fact that agents, such as laminin, sodium butyrate and dibutyryl cAMP, can induce the expression of neuronal, glial, and pigmented epithelial characteristics in vitro.
A human retinoblastoma cultured cell line, Y79 cells, when exposed to RPE-conditioned medium (RPE-CM), exhibits a high degree of neuronal-like differentiation. The differentiation is observed in both the morphological and biochemical characteristics of the cells. The exposed cells extend arborizing neuritic processes from their cell bodies and express elevated levels of neuron-specific enolase (NSE) and neurofilament proteins. In addition, RPE-CM extends the life of the attached, differentiated cells for more than 30 days as compared to 10 to 15 days for non-treated cells.
RPE-secreted proteins have been fractionated from RPE-CM, and a protein doublet, with an apparent molecular weight of about 50,000 to about 55,000, that is unique to RPE-CM has been identified. This pigment epithelium derived neurotrophic factor (PEDF), which is a major secretory product of human fetal RPE cells, has been isolated and shown to have neurotrophic effects on Y79 retinoblastoma cells. Additionally, a smaller 36,000 molecular weight, form of PEDF has also been identified.
PEDF has uses in the treatment of retinal diseases including, but not limited to, retinoblastoma and other ocular tumors, retinitis pigmentosa, various forms of retinal detachment, macular degeneration, diabetic retinopathy, and other inherited and age-related pathologies of retinal cells.
In the case of retinal tumors, PEDF induces the tumor cells to display biochemical and phenotypic characteristics of mature neuronal cells. Such changes are identified by a cessation or reduction in the rate of cell division, which leads to tumor regression or a slowing in the rate of tumor growth. In the case of non-tumorous retinal diseases, the neurotrophic properties of PEDF enhance survival and well-being of photoreceptor or other retinal cells, prolonging their functional life span and delaying the rate of the onset of impaired vision and ultimate blindness. In the case of retinal detachment, PEDF prolongs the life span of photoreceptor cells sufficiently to allow standard reattachment procedures to be effective in re-establishing the retina-RPE interface, thereby restoring normal vision.
1. Preparation of an Impure Protein Fraction Containing Retinal Pigmented Epithelium Derived Neurotrophic Factor
Retinal pigmented epithelium derived neurotrophic factor (PEDF) may be isolated from any tissue or cell producing PEDF.
Naturally-occurring cells that produce PEDF are retinal pigmented epithelium cells. Such cells may be grown in culture to secrete PEDF into the medium in which they are growing. The medium may be harvested periodically, and the PEDF isolated from the media. Suitable cell cultures may be established from retinal pigmented epithelium cells derived from humans, monkeys, and other primates or other animals, such as chickens, mice, rats, cows and pigs. PDEF may also be isolated from the vitreous humor of human, bovine, monkey and other primates. The vitreous contains an abundance of PEDF and is very easy to remove from the eye cup. This is probably the easiest source from which to isolate PEDF.
PEDF may also be isolated by extraction of the interphotoreceptor matrix (IPM) or from the retina of humans, monkeys, and other primates or other animals, such as chickens, mice, rats, cows and pigs.
Alternatively PEDF may be derived from sources in which it is not naturally-occurring, such as organisms transfected with a recombinant DNA molecule constructed to result in the expression of PEDF or a PEDF equivalent protein in the host cells chosen for the expression of the gene. It is well known in the art that proteins can be modified by deletion, addition or substitution of amino acids without changing the function of the protein. Such proteins which retain the specificity of PEDF are considered to be equivalent.
A. Culturing of Human Retinal Pigmented Epithelium (RPE) Cells
Cultures of RPE cells are established by harvesting post-mortem eyes by aseptically opening the eyes and removing the vitreous body and retina. The exposed RPE is washed with a buffered solution such as modified Earle""s balanced salt solution (MEBS: 115.5 mM NaCl, 3.5 mM KCl, 1 mM NaH2PO4, 0.5 mM CaCl2, 0.27 mM MgCl2, 0.37 MgSO4, 15 mM HEPES, 14 mM NaHCO3, 12 mM glucose, pH 7.2). RPE cells are scraped from Bruch""s membrane or are dislodged by a stream of fluid such as MEBS or cell culture medium, applied using a Pasteur or similar pipette. This step may be preceded by exposure to proteolytic or other enzyme(s), such as Dispase (Boehringer-Mannheim, Indianapolis, Ind., Catalog #295 825). Alternatively, RPE cells may be isolated by detaching sections of sclera from an intact eye to expose choroidal tissue and treating this choroidal surface with proteolytic or other enzymes such as Dispase prior to removal of the vitreous and retina and dislodging RPE cells by the spraying of cell culture medium as described by Pfeffer et al., J. Cell. Physiol. 117 333-341 (1983). Tissue fragments are transferred to tissue culture dishes in a medium such as xe2x80x9clow Ca++xe2x80x9d or xe2x80x9chigh Ca++xe2x80x9d complete RPE-47 medium, as described by Pfeffer et al., J. Cell. Physiol. 117 333-341 (1983), or Eagles""s minimal essential medium (MEM, supplied by GIBCO of Grand Island, N.Y.) supplemented with about 0.5% to about 20% v/v fetal calf serum. At concentrations below about 0.5% v/v fetal calf serum, the serum concentrations are too low to effectively support the growth of the cells, At concentrations above about 20% v/v serum, no additional benefit, with respect to cell growth, is conferred on the cells.
The medium may also be supplemented with antibiotics and/or fungicides to prevent the growth of bacteria and/or fungi in the cultures. Antibiotics and fungicides suitable for use in the present invention are about 1,000 units/ml penicillin, about 100 xcexcg/ml streptomycin, about 0.25 xcexcg amphotericin, and about 50 xcexcg/ml gentamicin, or other suitable such agents known in the art. These antibiotics may be used individually or in combination, as desired.
The cells are incubated at about 37xc2x0 C. in an atmosphere of about 5% v/v C2. Retinal pigmented epithelium (RPE) cells attach to the surface of the dishes, proliferate, and eventually form confluent monolayers.
When the cells have grown to confluence, about 3-7 days from the initial culturing of the cells, they are harvested, for example, by trypsinizing the cell monolayer, or by other methods known to those skilled in the art, and resuspending the cells in a medium such as MEM, supplied by GIBCO, supplemented with about 5% v/v to about 20% v/v fetal calf serum. A portion of the cells are reseeded into sterile tissue culture flasks, after which time the cells are again grown in an atmosphere of about 5% v/v CO2 at about 37xc2x0 C.
Alternatively, RPE cells may be isolated by removing the cornea, the 2 mm scleral ring, and the vitreous and neural retina from the eyes. The eyes are washed with calcium and magnesium-free Hanks Balanced Salt Solution (HBSS: 1.3 mM CaCl2, 5 mM KCl, 0.3 mM KH2PO4, 0.5 mM MgCl2, 0.4 mM MgSO4, 138 mM NaCl, 4 mM NaHCO3, 0.3 mM Na2HPO4, 5.6 mM D-glucose and 0.03 mM Phenol Red, supplied by GIBCO/BRL of Gaithersburg, Md.). The eye cup is filled with a solution comprising about 0.1% w/v trypsin, about 0.1% w/v hyaluronidase in calcium and magnesium-free Hanks balanced salt solution, and the eye is incubated at about 37xc2x0 C. for about 15 to about 30 minutes.
The loose RPE cells are collected by gentle aspiration, and the procedure is repeated until the RPE cells are released. The trypsin is inactivated by adding about 5% v/v to about 20% v/v fetal calf serum to the cell sample. The cells are collected by centrifugation at about 1,200 rpm for about 7 minutes.
The RPE cells are then plated onto sterile tissue culture plates at a density of about 1xc3x97105 cells for a 35 mm plate. (Proportionally more or less cells are plated if larger or smaller plates or containers are used.) The cells are grown in DulBecco""s Modified Eagles Medium (DMEM), supplied by GIBCO, or other suitable medium. The medium may be supplemented with an equal volume of HAM""s F12 medium (supplied by GIBCO), xe2x80x94about 1 mM sodium pyruvate, about 0.625 mM Hepes, about 6 mM L-glutamine, about 1% w/v non-essential amino acids, about 5 xcexcg/ml insulin, about 5 xcexcg/ml transferrin, about 5 ng/ml selenium, antibiotics as described above, and about 0.5% v/v to about 20% v/v fetal calf serum, as described above. The insulin, transferrin, and selenium are supplied by Collaborative Research of Lexington, Mass. Other reagents suitable for use in the present invention, unless otherwise specified, are supplied by Sigma Chemical Co. of St Louis, Mo.
When the cells have grown to confluence (about 3-7 days from the initial culturing of the cells), they are harvested, for example, by trypsinizing the cell monolayer or by other methods known to those skilled in the art, and resuspending the cells in a medium such as MEM supplemented with about 0.5% v/v to about 20% v/v fetal calf serum. A portion of the cells are reseeded into sterile tissue culture flasks, after which time the cells are again grown in an atmosphere of about 5% v/v CO2 at about 37xc2x0 C.
While only two methods for the culturing of RPE cells are described, other suitable methods for culturing RPE cells are known in the art. Such methods are also suitable for use in the practice of the present invention.
B. Preparation of Retinal Pigmented Epithelium Conditioned Medium (RPE-CM)
PEDF is a secreted protein, and RPE cells secrete PEDF into the medium in which they are grown. Therefore, a convenient method of producing PEDF is to grow RPE cells in culture and to periodically harvest the media from the cultures.
A method suitable for use in the present invention for isolating PEDF from medium is to allow RPE cells to grow to confluence in a medium such as DMEM supplemented with about 1 mM sodium pyruvate, about 0.625 mM Hepes, about 6 mM L-glutamine, about 1% w/v non-essential amino acids, about 5 xcexcg/ml insulin, about 5 xcexcg/ml transferrin, about 5 ng/ml selenium, antibiotics and/or fungicides as described above, and about 0.5% v/v to about 20% v/v fetal calf serum, as described above. The confluent cultures of RPE cells are washed extensively with Hank""s balanced salt solution, or other suitable wash solutions, to remove serum proteins derived from the fetal calf serum present in the media used to culture the RPE cells. About 1 ml, per cm2 of cell surface area, of serum-free medium such as DMEM supplemented with about 1 mM sodium pyruvate, about 0.625 mM Hepes, about 6 mM L-glutamine about 1% w/v non-essential amino acids, about 5 xcexcg/ml insulin, about 5 xcexcg/ml transferrin, about 5 ng/ml selenium, and antibiotics and/or fungicides as described above, is added to the cultures, and they are incubated in an atmosphere of about 5% v/v CO2 at about 37xc2x0 C. for about 1 to about 10 days. Alternatively, PEDF can be isolated from serum-containing medium such as DMEM supplemented with about 1 mM sodium pyruvate, about 0.625 mM Hepes, about 6 mM L-glutamine, about 1% w/v non-essential amino acids, about 5 xcexcg/ml insulin, about 5 xcexcg/ml transferrin, about 5 ng/ml selenium, antibiotics and/or fungicides as described above, and about 0.5% v/v to about 20% v/v fetal calf serum as described above.
The medium is then collected by pouring the medium into plastic centrifuge tubes, and the medium is centrifuged at about 3,000 rpm for about 10 minutes to remove any free cells and other particulate matter from the medium, and filtered to provide an impure PEDF protein solution. The medium may be used directly for the purification or testing of PEDF or it may be stored at xe2x88x9220xc2x0 C. until required.
C. Isolation of PEDF from Tissue Samples
PEDF may be isolated directly from eyes by opening the eyes and removing the vitreous body and retina. The retinal pigmented epithelium is scraped off the Bruch""s membrane using a disposable cell scraper. Tissue fragments are transferred to a Teflon or glass homogenizer in a solution such as 10 mM phosphate-buffered saline (PBS) and homogenized to break the cells. The solution is then centrifuged at about 10,000 rpm for about 10 minutes and filtered to remove cell debris. The supernatant, which contains PEDF, is collected to provide an impure PEDF protein solution. The medium may be used directly for the purification or testing of PEDF or it may be stored at xe2x88x9220xc2x0 C. until required.
D. Isolation of PEDF from Bovine Vitreous
The vitreous is a gel like substance which fills the eye cup. The vitreaous is removed from the eye and solubilized by freezing and thawing. About 20 ml of solubilized vitreous are obtained per bovine eye cup and this volume contains about 16 xcexcl/100 xcexcl of PEDF.
F. Isolation of PEDF from Recombinant Cells
PEDF may also be isolated from recombinant cells which have been constructed to express the PEDF gene. The PEDF may be expressed as an intracellular or an extracellular protein.
Intracellular PEDF is isolated by homogenizing the cells in a Dounce or other suitable homogenizer in a buffer, such as PBS, that may contain detergents or other solubilizing agents such as urea or guanidine hydrochloride, and centrifuging at about 10,000 rpm for about 20 minutes to remove cellular debris, to provide an impure PEDF protein fraction.
Extracellular PEDF is isolated by collecting the medium in which cells expressing PEDF are grown. This is most conveniently performed in a continuous centrifugation process, such as with a Sharples centrifuge. The supernatant is collected to provide an impure PEDF protein fraction. The medium may be used directly for the purification or testing of PEDF or it may be stored at xe2x88x9220xc2x0 C. until required.
2. Purification of PEDF
A. Small Scale Purification of PEDF
i. Ammonium Sulfate Precipitation
An impure PEDF protein fraction may be partially purified by ammonium sulfate precipitation to provide an ammonium sulfate purified PEDF protein fraction. Percent ammonium sulfate refers to % saturation of ammonium sulfate at 20xc2x0 C. and is based on a 100% saturation of 767 g/l.
An impure PEDF protein fraction is brought to about 50% saturation with ammonium sulfate by the addition of about 313 g of solid ammonium sulfate per liter of impure protein fraction at about 20xc2x0 C. The ammonium sulfate is preferably added slowly to the impure protein fraction while the solution is stirred, such as with a stir bar on a mechanical stirrer. The ammonium sulfate is added slowly to prevent localized high concentrations of ammonium sulfate that may result in rapid precipitation and denaturation of proteins present in the impure protein fraction. After all the ammonium sulfate is added, the about-50% ammonium sulfate solution is stirred for about 30 minutes at about 20xc2x0 C. The about-50% ammonium sulfate solution is then centrifuged at about 10,000 g, at about 20xc2x0 C. for about 20 minutes. The supernatant is collected for further processing. Alternatively, the about-50% ammonium sulfate solution may be filtered through filter paper such as Whatman #1, to remove the precipitate. In this case, the filtrate is collected for further processing.
The about-50% ammonium sulfate solution is then brought to about 70% saturation by the addition of about 137 g/l of ammonium sulfate. The ammonium sulfate is added slowly, and after all the ammonium sulfate is added, the about-70% ammonium sulfate solution is stirred for about 30 minutes at about 20xc2x0 C. The about-70% ammonium sulfate solution is centrifuged or filtered, as described above, and the precipitate is collected.
The ammonium sulfate precipitate is then redissolved in a buffer such as about 10 mM phosphate, pH 7, to form a 50-70% ammonium sulfate fraction. The solution is then diafiltered using an Amicon Diaflo ultrafiltration unit, or dialyzed against a buffer such as about 10 mM phosphate, pH 7, to remove the residual ammonium sulfate from the redissolved 50%-70% ammonium sulfate fraction.
ii. Purification of PEDF by SDS Gel Electrophoresis
A small-scale isolation of PEDF is conducted by SDS-polyacrylamide slab gels. Such polyacrylamide gels are well known in the art, and the preparation of such gels has been described by Weber and Osborn, J. Biol. Chem. 244 4406 (1969), as modified by Laemmli, Nature 277 680 (1970).
Samples of an impure PEDF protein fraction or ammonium sulfate purified PEDF protein fraction are dialyzed against a buffer such as about 10 nM sodium phosphate buffer, pH 7.5, lyophilized and resuspended in 62.5 mM Tris, pH 6.8, 2% w/v SDS, 10% v/v glycerol, 0.001% w/v bromophenol blue, and 0.1 M 2-mercaptoethanol, wherein the bromophenol blue is a migration marker. Additional samples to be loaded on the gel include molecular-weight standards such as phosphorylase B, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor and lysozyme (such as supplied by Bio-Rad, catalog #161-0304), and controls as may be necessary. In cases where conditioned media are used as samples, media which have not been exposed to RPE cells (unconditioned media) are included as controls.
The samples are then incubated in a boiling-water bath for about 5 minutes and loaded onto SDS-polyacrylamide slab gels. The markers and unconditioned media are loaded into wells on the outside of the gel, and the impure PEDF protein fractions or the ammonium sulfate purified PEDF protein fraction are loaded on the remaining inside wells. The samples are then subjected to electrophoresis. The electrophoresis is continued until the bromphenol blue marker has reached the bottom of the gel. At the completion of electrophoresis, strips from each outside edge of the gels, which included the markers and a lane each of an impure PEDF protein fraction and an unconditioned media control, are stained with Coomassie blue. The stained protein bands which develop on the stained strips are aligned with the unstained portion of the gel to locate the position of the proteins present in the impure PEDF protein fraction or the ammonium sulfate purified PEDF protein fraction but absent from control media.
A PEDF protein doublet, with an apparent molecular weight of about 50,000 to about 55,000, unique to the impure PEDF protein fraction, is excised and the proteins electro-eluted, by methods known in the art, from the unstained portion of the gel. The eluant is centrifuged to remove gel fragments and the supernatant dialyzed against 10 mM phosphate-buffered saline (145 mM NaCl, 8.1 mM Na2HPO4, and 1.9 mM NAH2PO4.H2O) to provide an SDS-polyacrylamide purified PEDF protein fraction.
iii. Purification of PEDF Using BioRad PREP CELL
Large quantities of PEDF can be purified by using BioRad PREP CELL which is an electrophoresis unit containing a tube gel. Samples are loaded onto the top of the tube gel and fractions are collected from the bottom. Individual fractions are examined by SDS gel electrophoresis for a 50,000 molecular weight protein. Fractions containing a 50,000 molecular weight protein are collected and pooled.
iv. Purification of PEDF by Cation-Exchange HPLC
An impure PEDF protein fraction or an ammonium sulfate purified PEDF protein fraction is dialyzed against water or a buffer such as about 10 mM phosphate buffer, pH 7.2, or other suitable buffer, to remove media and salts from the impure protein sample. PEDF may be purified by cation-exchange HPLC using a column chromatography medium, such as that supplied under the trade name xe2x80x9cBrownlee Aquapore CX-300xe2x80x9d by Western Analytical, Temecula, Calif., packed into a column, such as a 4.6xc3x9730 mm column, or other suitable cation-exchange HPLC chromatography medium.
The chromatography medium is equilibrated with a buffer such as about 10 mM phosphate, pH 7.2. The dialyzed impure PEDF protein fraction or ammonium sulfate purified PEDF protein fraction is loaded onto the chromatography medium, and the chromatography medium with PEDF bound to it is washed with a buffer such as about 10 mM phosphate, pH 7.2, until all unbound proteins are washed from the chromatography medium. PEDF is eluted from the chromatograph medium with a linear salt gradient from about 0.0 to about 0.5 M NaCl. PEDF elutes as a single peak with a NaCl concentration of about 0.25 M.
The eluted PEDF is concentrated by lyophilization and resolubilized in water or a buffer such as about 10 mM phosphate buffer, pH 7.2, or other suitable buffer, to form a cation-exchange HPLC purified PEDF protein fraction.
iv. Purification of PEDF by Reverse-Phase HPLC
An impure PEDF protein fraction or ammonium sulfate purified PEDF protein fraction is dialyzed against a buffer such as about 10 mM phosphate buffer, pH 7.2, or other suitable buffer, to remove media and salts from the impure protein sample. PEDF may be purified by reverse-phase HPLC using a column chromatography medium such as a chromatography medium supplied under the trade name xe2x80x9cVydac C8xe2x80x9d by The Separation Group of Hesperia, Calif., packed into a column such as a 4.6xc3x97250 mm column or other suitable reverse-phase HPLC chromatography medium.
The chromatography medium is equilibrated with a solution such as about 0.1% v/v trifluoroacetic acid (TFA). The dialyzed impure PEDF protein fraction or ammonium sulfate purified PEDF protein fraction is loaded onto the chromatography medium, and the chromatography medium with PEDF bound to it is washed with an eluant such as 0.1% v/v TFA, until all unbound proteins are washed from the chromatography medium. PEDF is eluted from the chromatograph medium with a linear gradient from about 0.1% v/v TFA in water to about 95% v/v acetonitrile (CH3CN), 0.1% v/v TFA, 5% v/v H2O. PEDF elutes as a single peak with a CH3CN concentration of about 70% v/v.
The eluted PEDF is concentrated by lyophilization and resolubilized in water or a buffer such as about 10 mM phosphate buffer, pH 7.2, or other suitable buffer, to form a reverse-phase HPLC purified PEDF protein fraction.
V. Size-Exclusion HPLC
An impure PEDF protein fraction, an ammonium sulfate purified PEDF protein fraction, a cation-exchange HPLC purified PEDF protein fraction, or a reverse-phase HPLC purified PEDF protein fraction may be purified by size-exclusion chromatograph using a chromatography medium, such as that supplied under the trade name xe2x80x9cBio-Rad TSK-250xe2x80x9d by Bio-Rad of Richmond, Calif., packed into a column such as a 7.5xc3x97300 mm column. The impure PEDF protein fraction, the ammonium sulfate purified PEDF protein fraction, the cation-exchange HPLC purified PEDF protein fraction, or the reverse-phase HPLC purified PEDF protein fraction is loaded onto the size-exclusion chromatography medium, which has been equilibrated with a buffer such as 0.02 M Tris-HCl, pH 7.0, 0.6 M NaCl. PEDF-containing fractions are collected and dialyzed against a buffer such as about 10 mM phosphate buffer, pH 7.2 to provide a size-exclusion purified PEDF protein fraction.
vi. Heparin Chromatography
An impure PEDF protein fraction or an ammonium sulfate purified PEDF protein fraction may also be purified by heparin chromatography. A heparin chromatography medium such as heparin agarose, supplied by Sigma Chemical Co. of St Louis, Mo. (Cat. No. H-5380), is equilibrated with a buffer such as about 10 mM Tris-HCl, pH 7.5.
An impure PEDF protein fraction or an ammonium sulfate purified PEDF protein fraction is dialyzed against a buffer such as about 10 mM Tris, pH 7.5, to remove any salts or media from the samples, and the dialyzed PEDF solution is applied to the equilibrated heparin agarose. After the PEDF solution has been applied to the heparin agarose, the heparin agarose is washed with a buffer such as about 10 mM Tris-HCl, pH 7.5, until all unbound proteins are eluted from the heparin agarose. PEDF is then eluted from the heparin agarose with a buffer such as about 10 mM Tris-HCl, pH 7.5, 0.5 M NaCl.
The eluate containing PEDF is then diafiltered in an Amicon Diaflo ultrafiltration unit, or is dialyzed against a buffer such as about 10 mM Tris-HCl, pH 7.5, to remove NaCl present in the eluate, thereby providing a heparin purified PEDF protein fraction.
The above-described purification procedures may use an impure PEDF protein fraction or an ammonium sulfate purified PEDF protein fraction as the starting material for the subsequent column purification. Alternatively, a protein fraction which has already been purified by one or more of the described chromatography steps could also be used. Therefore, the purification procedures may be used alone or in combination with each other or with other purification techniques known in the art to produce a PEDF protein fraction of the desired purity.
B. Large-Scale Preparation of PEDF
i. Ammonium Sulfate Precipitation
Large-scale purification of PEDF may be prepared by ammonium sulfate precipitation to provide an ammonium sulfate purified PEDF protein fraction.
An impure PEDF protein fraction is brought to about 50% saturation with ammonium sulfate by the addition of about 313 g of solid ammonium sulfate per liter of impure PEDF protein fraction. The ammonium sulfate is preferably added slowly to the impure protein fraction while the solution is stirred, such as with a stir bar on a mechanical stirrer. The ammonium sulfate is added slowly to prevent localized high concentrations of ammonium sulfate which may result in rapid precipitation, and denaturation of proteins present in the impure protein fraction. After all the ammonium sulfate is added, the 50% ammonium sulfate solution is stirred for about 30 minutes at 20xc2x0 C. The 50% ammonium sulfate solution is then centrifuged at about 10,000 g, at 20xc2x0 C. for about 20 minutes. The supernatant is collected for further processing. Alternatively, the 50% ammonium sulfate solution may be filtered through filter paper, such as Whatman #1, to remove the precipitate. The filtrate is collected for further processing.
The 50% ammonium sulfate solution is then brought to about 70% saturation by the addition of about 137 g/l of ammonium sulfate. The ammonium sulfate is added slowly, and after all the ammonium sulfate is added, the 70% ammonium sulfate solution is stirred for about 30 minutes at 20xc2x0 C. The 70% ammonium sulfate solution is centrifuged or filtered, as described above, and the precipitate is collected.
The ammonium sulfate precipitate is then redissolved in a buffer such as about 10 mM phosphate, pH 7, to form a 50-70% ammonium sulphate fraction, then diafiltered using an Amicon Diaflo ultrafiltration unit, or dialyzed against about 10 mM sodium phosphate, pH 7, to remove the ammonium sulfate from the redissolved 50-70% fraction to provide an ammonium sulfate purified PEDF protein fraction.
ii. Anion-Exchange Chromatography
For use, an anion-exchange chromatography medium such as DEAE cellulose is equilibrated with a buffer such as about 10 mM Tris, pH 7.5. The PEDF is applied to the anion-exchange chromatography medium, and the medium is washed with a buffer such as about 10 mM Tris, pH 7.5, until all unbound proteins are eluted from the column. After all the unbound proteins are eluted, PEDF is eluted with a linear salt gradient from about 0 to about 1 M NaCl in a buffer such as about 10 mM Tris-HCl, pH 7.5. The fractions containing PEDF are collected and pooled. These fractions are then diafiltered or dialyzed against a buffer such as about 10 mM Tris-HCl, pH 7.5, to remove NaCl, thereby providing an anion-exchange chromatography purified PEDF protein fraction.
Anion-exchange chromatography may be conducted by either batch or column chromatography techniques.
iii. Heparin Chromatography
Heparin chromatography may also be used for the large-scale purification of PEDF. A heparin chromatography medium such as heparin agarose, supplied by Sigma Chemical Co. (Cat. No. H-5380), is equilibrated with a buffer such as about 10 mM Tris-HCl, pH 7.5.
An impure PEDF protein fraction or an ammonium sulfate purified PEDF protein fraction is dialyzed against a buffer such as about 10 mM Tris-HCl, pH 7.5, and then applied to the heparin agarose. After the PEDF solution has been applied to the heparin agarose, the heparin agarose is washed with a buffer such as about 10 mM Tris-HCl, pH 7.5, until all unbound proteins are eluted from the heparin agarose. PEDF is then eluted from the heparin agarose with a buffer such as about 10 mM Tris-HCl, pH 7.5, 0.5 M NaCl.
The eluant containing PEDF is collected and diafiltered in an Amicon Diaflo ultrafiltration unit, or dialyzed against a buffer such as about 10 mM Tris-HCl, pH 7.5, to remove the NaCl present in the eluate, to provide a heparin purified PEDF protein fraction.
The above-described purification procedures may use an impure PEDF protein fraction or an ammonium sulfate purified PEDF protein fraction as the starting material for the subsequent column purification. Alternatively, a protein fraction which has already been purified by one or more of the described chromatography steps could also be used. Therefore, the purification procedures may be used alone or in combination with each other or with other purification techniques known in the art to produce a PEDF protein fraction of the desired purity.
3. PEDF Assays
A. SDS Gel Electrophoresis
PEDF may be identified by SDS-polyacrylamide gel electrophoresis on, for example, 7.5%, 10%, 12.5% w/v SDS-polyacrylamide gels. The preparation of such gels has been described by Weber and Osborn J. Biol. Chem. 244 4406 (1969), as modified by Laemmli, Nature 277 680 (1970), and the preparation and use of such gels are well known in the art.
The PEDF protein samples are mixed with about 5 xcexcl of 62.5 mM Tris, pH 6.8, 12% w/v SDS, 0.001% w/v bromophenol blue, 10% v/v glycerol, and 0.1 M 2-mercaptoethanol, wherein the bromophenol blue is a marker. Molecular-weight marker samples which include molecular-weight standards such as phosphorylase B, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor and lysozyme (such as supplied by Bio-Rad, catalog #161-0304) are included on the gels as controls. The PEDF protein samples and the molecular-weight standards are boiled for about 5 minutes and loaded onto separate wells on the SDS-polyacrylamide slab gels. The samples are then subjected to electrophoresis until the bromphenol blue has migrated to the bottom of the gel. At the completion of electrophoresis, the gel is silver-stained, stained with Coomassie blue, or stained by other suitable protein staining methods. The molecular weight of proteins in the PEDF sample is then compared to the molecular-weight standards.
Alternatively, larger quantities of PEDF can be collected from SDS-PAGE tube gels using BioRad PREP CELL equipment and a fraction collector.
PEDF migrates as a protein doublet, with an apparent molecular weight of from about 50,000 to about 55,000 on SDS polyacrylamide gels.
B. Neuronal Inductivity
PEDF activity in protein samples may be assayed by its neuronal inductivity. Various concentrations of PEDF are added to cultures of cells such as Y79 retinoblastoma (RB) cells, supplied by the American Type Culture Collection, Access No. HTB 18, of Rockville, Md.
The Y79 RB cells are grown in suspension culture. The cells are harvested by centrifugation for about 5 minutes at about 900 rpm at room temperature and resuspended in a serum-free medium such as Dulbecco""s modified Eagle""s medium supplemented with 5 ug/ml insulin, 5 ug/ml transferrin, 5 ug/ml selenous acid, and 876.6 ug/ml L-glutamine (serum-free medium), which has previously been warmed to about 37xc2x0 C. The collected cells are resuspended at a concentration of about 106 cells/ml in serum-free medium. About 50 to about 500 ng/ml of PEDF is added to about 25 ml aliquots of the cells. The cells are incubated for about 7 days at about 37xc2x0 C., then attached to poly-D-lysine-coated flasks or glass coverslips. The poly-D-lysine-coated flasks are prepared by coating with a solution containing about 200 ug/ml poly-D-lysine (such as that supplied by Sigma, Catalog #P7405) for about 1-24 hours, followed by rinsing with water and serum-free medium. Other cells may be used and it will be clear to one skilled in the art that testing other cells is a routine matter of following the methods set out above with the desired cell line.
i. Cell Analyses
Morphology of attached cells is monitored daily by phase contrast microscopy of living cells using a microscope such as an inverted Diaphot TMD microscope, supplied by Nikon of Tokyo, Japan, or by differential interference contrast microscopy of cells, fixed with a fixative such as about 4% v/v paraformaldehyde in 0.1 M sodium cacodylate buffer, using a microscope, such as a BHS-BH2 microscope, supplied by Olympus of Tokyo, Japan.
Differentiation is assessed by calculating the percentage of cellular aggregates (more than 90% of Y79 cells plated from suspension culture attach to poly-D-lysine-coated flasks as aggregates containing more than 5 cells) in which cells extend processes at day 1, 3, 7 and 11 after attachment. Experiments are performed in replicates of 3 and are repeated twice.
Expression of neuron-specific enolase (NSE) and neurofilament 200,000 molecular weight protein subunit (NF 200) is monitored by immunofluorescence and viewed by either epifluorescence microscopy (Olympus BHS-BH2 microscope) or microspectrofluorometry (MSA, Farrand Microscope Spectrum Analyzer). Quantification is by 1) visual scoring of intensity of fluorescence at 485 nm excitation as +=weak; ++=moderate; +++=strong; ++++=very intense, and 2) microspectrofluorometric analysis (MSA) readings (xcexcAxc3x97100, time constant of about 0.3 seconds; specimen size of approximately 2 mm or about 100 cells/aggregate; target size of about 15 xcexcm or approximately that of 1 cell).
The presence of PEDF in the protein sample results in the Y79 RB cells, extending neurite-like processes. At a concentration of about 500 ng/ml, about 70% of the cells extend neurite-like processes.
ii. Differentiation
Cells were cultured as described above. The cells were then monitored daily by phase-contrast microscopy of living cells (Olympus IMT-2) and by differential interference contrast microscopy (Olympus BHS) of cells fixed with a fixative such as about 4% v/v paraformaldehyde. The percentage of differentiating cells is estimated by calculating the number of cellular aggregates containing five or more cells exhibiting neurite outgrowths following 8-10 days of culture on a poly-D-lysine substratum.
With about 50 to about 500 ng/ml PEDF, approximately 80% of the cells undergo morphological differentiation within about 3 days.
4. Characterization of the PEDF Protein
A. Isolation of PEDF Peptides
Purified PEDF, about 500 xcexcg, is concentrated using Centricon 10 microconcentrators (Amicon, Danvers, Mass.), and then diluted in a suitable digestion buffer such as about 25 mM Tris, pH 8.5, 1 mM EDTA. To the protein sample is added a proteolytic enzyme, such as endoproteinase Lys-C, supplied by Boehringer-Mannheim of Indianapolis, Ind. The PEDF/proteinase mixture is incubated for about 18 hours at about 30xc2x0 C., or until the reaction has gone to completion, i.e., until all the PEDF is completely digested by the proteinase. Alternatively, the protein may be digested with trypsin in a buffer comprising about 10 mM PBS or by using other proteinases known in the art.
The resulting PEDF polypeptide fragments are separated by using a separation system such as HPLC on a Vydac C8 reverse-phase column. A 4.6xc3x97250 mm column is suitable for use in the present invention. The column is equilibrated with about 0.1% v/v TFA in water. The polypeptides are eluted with about 90% v/v CH3CN, 0.1% v/v TFA and 5% v/v H2O.
Polypeptides eluted from the column which are well separated from other polypeptides are collected and subjected to protein sequencing analysis.
B. Protein Sequencing Analysis
The purified polypeptide fragments are subjected to amino-acid sequence analysis by methods known in the art. Alternatively, the amino-acid sequence analysis is conveniently performed under contract at an amino-acid sequencing facility, such as the Microsequencing Facility of Beckman Research Institute at the City of Hope in Duarte, Calif.
5. Characterization of the PEDF Gene
The present invention provides, among other things, a nucleic acid which encodes PEDF. In particular, a cDNA sequence is provided for human PEDF as set forth in SEQ ID NO:1, for mouse PEDF as set forth in SEQ ID NO:7 and for human PEDF as set forth in SEQ ID NO:8. This cDNA sequence codes for PEDF, which has the amino acid sequence set forth in SEQ ID NO:2. The cDNA and amino acid sequences are listed in the GenBank(copyright) Data Bank under accession number M76979.
The term xe2x80x9cnucleic acidxe2x80x9d refers to a polymer of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), which can be derived from any source, can be single- or double-stranded, and can optionally contain synthetic, non-natural, or altered nucleotides which are capable of being incorporated into DNA or RNA polymers. The nucleic acid of the present invention is preferably a segment of DNA.
The present invention further provides a truncated version of PEDF, which is referred to as PEDF-BH. PEDF-BH comprises the amino acid sequence Met-Asn-Arg-Ile fused to Asp44 . . . Pro418 of PEDF, the amino terminus of which has been deleted. The truncated protein comprises the amino acid sequence of SEQ ID NO:3. The present invention also provides a nucleic acid which encodes a protein comprising the amino acid sequence of PEDF-BH, i.e., the amino acid sequence of SEQ ID NO:3.
One who is skilled in the art will appreciate that more than one nucleic acid may encode any given protein in view of the degeneracy of the genetic code and the allowance of exceptions to classical base pairing in the third position of the codon, as given by the so-called xe2x80x9cWobble rules.xe2x80x9d Moreover, nucleic acids that include more or less nucleotides can result in the same or equivalent proteins. Accordingly, it is intended that the present invention encompass all nucleic acids that encode the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:3, as well as equivalent proteins. The phrase xe2x80x9cequivalent nucleic acidsxe2x80x9d is intended to encompass all of these nucleic acids.
It also will be appreciated by one skilled in the art that amino acid sequences may be altered without adversely affecting the function of a particular protein. In fact, some alterations in amino acid sequence may result in a protein with improved characteristics. The determination of which amino acids may be altered without adversely affecting the function of a protein is well within the ordinary skill in the art. Moreover, proteins that include more or less amino acids can result in proteins that are functionally equivalent. Accordingly, it is intended that the present invention encompass all amino acid sequences that result in the PEDF and PEDF-BH proteins, proteins that are functionally equivalent to PEDF and PEDF-BH, and proteins derived therefrom. The phrase xe2x80x9cequivalent proteinsxe2x80x9d is intended to encompass all of these amino acid sequences.
Some examples of possible equivalent nucleic acids and equivalent proteins include nucleic acids with substitutions, additions, or deletions which direct the synthesis of the PEDF and PEDF-BH proteins and equivalent proteins; nucleic acids with different regulatory sequences that direct the production of the PEDF and PEDF-BH proteins; variants of PEDF-BH which possess different amino acids and/or a number of amino acids other than four fused to the amino terminal end of the protein; and PEDF and PEDF-BH proteins with amino acid substitutions, additions, deletions, modifications, and/or posttranslational modifications, such as glycosylations, that do not adversely affect activity.
The present invention also provides a vector which comprises a nucleic acid of SEQ ID NO:1, a nucleic acid which encodes a protein comprising the amino acid sequence of SEQ ID NO:2 or an equivalent protein, a nucleic acid which encodes a protein comprising the amino acid sequence of SEQ ID NO:3 or an equivalent protein, and equivalent nucleic acids thereof.
A. Cloning of the PEDF cDNA
Oligonucleotides are constructed from the sequence derived for the isolated polypeptide of PEDF on an ABI 392 DNA/RNA Synthesizer or by methods well known in the art.
The oligonucleotides are used as primers for a polymerase chain reaction (PCR) by using a Techne thermal cycler and standard reagents and methodologies, supplied under the trade name xe2x80x9cGeneAMPxe2x80x9d by Perkin-Elmer/Cetus of Emeryville, Calif.
A human fetal eye Charon BS CDNA library is screened by PCR techniques using a Techne thermal cycler and standard reagents and methodologies. The cDNA fragment generated by the reaction is isolated on a 3% w/v NuSieve 3:1 gel (FMC Biochemicals) using NA-45 DEAE-cellulose paper (Schleicher and Schull) as described by Sambrook et al., 1989 In: Molecular Cloning: A Laboratory Manual 2nd ed. Cold Spring Harbor Press, Cold Spring Harbor, N.Y. Briefly, the screening procedure is performed by taking about 1, 5 and 50 xcexcl aliquots of the library and placing them in 600 xcexcl siliconized reaction tubes and then bringing them to a final volume of about 74 xcexcl with double-distilled sterile water. The phage particles are disrupted by incubation at about 70xc2x0 C. for about 5 minutes and then cooling on wet ice.
PCR master mix is made up in a 600 xcexcl reaction tube for 3 reaction tubes as follows:
30 xcexcl of 10xc3x97 Taq polymerase buffer;
24 xcexcl of dNTP mix; and an appropriate volume of double-distilled water is added to bring the master mix to a final volume of about 78 xcexcl.
The final solution, therefore, comprises about 192 mM KCl, 38.5 mM Tris-HCl, pH 8.3, 51.8 mM MgCl2, 0.038% w/v gelatin, 0.77 mM of each dNTP, and 3.8 xcexcl of each oligonucleotide primer. About 26 xcexcl of master mix is added to each reaction tube. The library aliquots and the master mix solutions are overlayed with about 100 xcexcl of mineral oil and heated to about 94xc2x0 C. for about 5 minutes. The solutions are then equilibrated to the desired primer annealing temperature.
After the solutions have been equilibrated to the desired primer annealing temperature, about 1.5 xcexcl of Taq polymerase is added to the solution and incubated for about 20 minutes.
Thermal cycling is continued by incubating: at about 72xc2x0 C. for about 3 minutes for primer extension, however, this primer extension time may be varied if desired; at about 94xc2x0 C. for about 1 minutes about 20 seconds to denature the extension product from the template; at about 37xc2x0 C. for about 2 minutes to anneal the primers to a template; and at about 72xc2x0 C. for about 3 minutes for primer extension. The xe2x80x9ccyclexe2x80x9d is the repeated for a total of about 25 to about 30 cycles. At the end of the last cycle, a final primer extension step, of about 7 minutes at 72xc2x0 C. is added.
The fragment is labelled with 32p by random priming using a kit supplied under the trade name xe2x80x9cPrime-It Random Primer Labeling Kitxe2x80x9d supplied by Stratagene Inc., La Jolla, Calif. The labelled probe is used to screen about 200,000 plaque-forming units of the Charon BS cDNA library by methods well known in the art.
Positive clones are isolated, and the DNA purified, with reagents supplied under the trade name xe2x80x9cQiagen Maxixe2x80x9d by Qiagen Inc. of Studio City, Calif.
The inserts within the phage vector are excised with an appropriate restriction enzyme, circularized with T4 DNA ligase supplied by New England Biolabs of Beverly, Mass. and transformed into competent E. coli Sure cells supplied by Stratagene. The cells are then plated on ampicillin/5-bromo-4-chloro-3-indolyl-xcex2-D-galactoside (X-gal) plates.
White colonies are selected and mini-prepped using a plasmid miniprep reagent supplied by Qiagen. Purified plasmids are digested to excise the insert, and the fragments are separated by gel electrophoresis to determine the size of the inserts. A clone with an appropriately-sized insert is then chosen for further investigation.
The cDNA""s for mouse and bovine PEDF were obtained following similar procedures.
B. Cloning of the Genomic DNA Sequence
The cDNA insert is labeled ith xcex132P dCTP and used to screen two genomic DNA libraries: a cosmid library constructed from MboI partial digests of human placental DNA (Clonetech) and a human placental genomic library constructed in xcex DASH II (Stratagene). Positively hybridizing clones are analyzed by Southern blot analysis. Two strongly hybridizing fragments: a 7.1 kb BamHI fragment from the cosmid clone and a 7.2 kb Not1 fragment from the xcex DASH II clone are selected for subcloning and DNA sequencing.
In an alternative embodiment of the present invention, primers, designed from the internal coding region of the PEDF cDNA sequence are synthesized using an ABI 392 DNA/RNA synthesizer and used as primers in polymerase chain reaction (PCR) experiments. The primer sequences are as follows: primer 603:
5xe2x80x2-ACAAGCTGGC AGCGGCTGTC-3xe2x80x2 (SEQ ID NO:9) is located in SEQ ID NO:1 at 271-290; primer 604:
5xe2x80x2-CAGAGGTGCC ACAAAGCTGG-3xe2x80x2 (SEQ ID NO:10) is located on the antisense strand in SEQ ID NO:1 at 570-590; primer 605:
5xe2x80x2-CCAGCTTTGT GGCACCTCTG-3xe2x80x2 (SEQ ID NO:11) is located in SEQ ID NO:1 at 570-590; primer 606:
5xe2x80x2-CATCATGGGG ACCCTCACGG-3xe2x80x2 (SEQ ID NO:12) is located on the antisense strand in SEQ ID NO:1 at 829-848; primer 2213:
5xe2x80x2-AGGATGCAGG CCCTGGTGCT-3xe2x80x2 (SEQ ID NO:13) is located in SEQ ID NO:1 at 114-133; primer 2744:
5xe2x80x2-CCTCCTCCAC CAGCGCCCCT-3xe2x80x2 (SEQ ID NO:14) is located on the antisense strand in SEQ ID NO:1 at 224-244; primer 2238:
5xe2x80x2-ATGTCGGACC CTAAGGCTGT T-3xe2x80x2 (SEQ ID NO:15) is located in SEQ ID NO:1 at 846-866; primer 354:
5xe2x80x2-TGGGGACAGT GAGGACCGCC-3xe2x80x2 (SEQ ID NO:16) is located on the antisense strand in SEQ ID NO:1 at 1043-1062. The primer pairs 603:604 amplified a single 2 kb PCR product (jt108); 605:606 amplified a single 3.3 kb PCR product (jt109); 2213:2744 amplified a single 2.3 kb PCR product (jt115) and 2238:354 amplified a single 1.5 kb PCR product (jt116).
In an alternative embodiment of the present invention, two sets of primers JT10-UP01:JT10-DP01 corresponding to bases 6536-6559 of jt106 genomic sequence and 1590:1591 corresponding to bases 1-89 of SEQ ID NO:1 are used in PCR reactions to isolate P1 clones (Genome Systems). The primer sequences are as follows: primer JT10-UP01:
5xe2x80x2-GGTGTGCAAA TGTGTGCGCC TTAG-3xe2x80x2 (SEQ ID NO:17) is located in SEQ ID NO: 4 at 6536; primer JT10-DP01:
5xe2x80x2-GGGAGCTGCT TTACCTGTGG ATAC-3xe2x80x2 (SEQ ID NO:18) is located in SEQ ID NO:4 at 7175; primer 1590:
5xe2x80x2-GCACGCTGGA TTAGAAGGCA GCAAA-3xe2x80x2 (SEQ ID NO:19) is located is SEQ ID NO:1 at 1-25; and primer 1591:
5xe2x80x2-CCACACCCAG CCTAGTCCC-3xe2x80x2 (SEQ ID NO:20) is located in SEQ ID NO:1 at 71-89.
C. DNA Sequence Analysis
Sequence analysis is performed with an automated sequencer or by other techniques well known in the art.
6. Expression of the PEDF Gene in Recombinant Cells
Commercial or large-scale production of PEDF may be achieved by expression of the gene, after cloning into an appropriate vector, in a suitable host cell. One such suitable vector/host system is the baculovirus/insect cell systems.
In one embodiment of the present invention, the PEDF gene is cloned into a baculovirus transfer vector such as pAC373, described by Lithgow et al., DNA and Cell Biology 10 443-449 (1991); pVL941, described by Ghiasi et al., Virology 185 187-194 (1991); or other such baculovirus transfer vectors that are well known by those skilled in the art.
Generally, such vectors comprise the polyhedron promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV), inserted into a transfer vector such as pAc373, described by Summers and Smith (A manual for baculovirus vector and insect cell culture procedures, Texas Agric. Exp. Station Bull. No. 1555). Recombinant plasmids are prepared by methods well known in the art, such as those described by Maniatis et al. In: Molecular Cloning: A Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982).
The host cells, such as S. frugiperda (Sf9), are grown in TC100 medium supplied by GIBCO, supplemented with 10% v/v fetal calf serum. The Sf9 cells are co-transfected with purified infectious AcNPV DNA, about 1 xcexcg, and about 50 xcexcg of the recombinant DNA. Culture supernatants are harvested about 4 days after transfection. Recombinant viruses are identified by plaque hybridization or radiolabelling of the proteins produced.
The above description provides an example of the expression of the PEDF gene in a vector/host expression system. Other expression systems are known in the art; for example, Saccharomyces cerevisiae has been used in conjunction with a number of vectors such as those described by Silar et al., Gene 104 99-102 (1991), Dietrich et al., Eur. J. Biochem. 201 399-407 (1991), or Akiyoshi-Shibata et al., DNA and Cell Biology 10 613-612 (1991); recombinant vaccinia virus vectors in HeLa host cells such as those described by Nakano et al., Gene 105 173-178 (1991). Any such methods or other methods known in the art are suitable for use in the present invention for expression of the PEDF gene.
The PEDF gene may be expressed as a transported protein where the PEDF is isolated from the medium in which the recombinant expression host is grown, or may be expressed as an intracellular protein by deleting the leader or other peptides, in which case the PEDF is isolated from the host cells. The PEDF so isolated is then purified by the methods described above.
In another embodiment of the present invention the vector xcfx80FS 17, ATCC Deposit No. PTA-2753, which comprises the nucleic acid of SEQ ID NO:1, and the vector pEV-BH which comprises a nucleic acid which encodes a protein comprising the amino acid sequence of SEQ ID NO:3. It will be appreciated by those skilled in the art that the cDNA inserts described can be present in alternative vectors. For example, inserts can be in vectors of different nature, such as phages, viral capsids, plasmids, cosmids, phagemids, YACs, or even attached to the outside of a phage or viral capsid. The vectors can differ in host range, stability, replication, and maintenance. Moreover, the vectors can differ in the types of control exerted over cloned inserts. For example, vectors can place cloned inserts under the control of a different promoter, enhancer, or ribosome binding site, or even organize it as part of a transposon or mobile genetic element.
The present invention also provides a host cell into which a vector, which comprises a nucleic acid of SEQ ID NO:1, a nucleic acid which encodes a protein comprising the amino acid sequence of SEQ ID NO:2 or an equivalent protein, a nucleic acid which encodes a protein comprising the amino acid of SEQ ID NO:3 or an equivalent protein, or an equivalent nucleic acid thereof, has been introduced. In particular, the host cell may have the vector xcfx80FS17, which comprises the nucleic acid of SEQ ID NO:1, or the vector pEV-BH, which comprises a nucleic acid which encodes a protein comprising the amino acid sequence of SEQ ID NO:3.
The vectors of the present invention can be introduced into any suitable host cell, whether eukaryotic or prokaryotic. These host cells may differ in their preferred conditions for growth, their nutritive requirements, and their sensitivity to environmental agents. Any appropriate means of introducing the vectors into the host cells may be employed. In the case of prokaryotic cells, vector introduction may be accomplished, for example, by electroporation, transformation, transduction, conjugation, or mobilization. For eukaryotic cells, vectors may be introduced through the use of, for example, electroporation, transfection, infection, DNA coated microprojectiles, or protoplast fusion.
The form of the introduced nucleic acid may vary with the method used to introduce the vector into a host cell. For example, the nucleic acid may be closed circular, nicked, or linearized, depending upon whether the vector is to be maintained as an autonomously replicating element, integrated as provirus or prophage, transiently transfected, transiently infected as with a replication-disabled virus or phage, or stably introduced through single or double crossover recombination events.
The present invention also provides a method of producing PEDF, PEDF-BH, and equivalent proteins, which method comprises expressing the protein in a host cell. For example, a host cell into which has been introduced a vector which comprises a nucleic acid of SEQ ID NO:1, a nucleic acid which encodes a protein comprising the amino acid sequence of SEQ ID NO:2 or an equivalent protein, a nucleic acid which encodes a protein comprising the amino acid of SEQ ID NO:3 or an equivalent protein, or an equivalent nucleic acid thereof, may be cultured under suitable conditions to produce the desired protein. In particular, a host cell into which has been introduced the vector xcfx80FS17, which comprises the nucleic acid of SEQ ID NO:1, or the vector pEV-BH, which comprises a nucleic acid which encodes a protein comprising the amino acid sequence of SEQ ID NO:3, may be cultured under suitable conditions to produce the proteins comprising the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:3, respectively.
The present invention also provides recombinantly produced PEDF, PEDF-BH, and equivalent proteins, which have been produced in accordance with the aforementioned present inventive method of culturing an appropriate host cell to produce the desired protein. The production of a protein such as PEDF by recombinant means enables the obtention of large quantities of the protein in a highly purified state, free from any disease-causing agents which may accompany the protein isolated or purified from a naturally occurring source organism, and obviates the need to use, for example, fetal tissue as a source for such a protein.
The provision of cDNA sequences in the present invention also enables the obtention of human genomic DNA sequences that encode PEDF or that have substantial sequence homology to PEDF-BH.
The obtention of genomic DNA sequences from cDNA sequences can be accomplished using standard techniques.
Recombinant PEDF, PEDF-BH, and equivalent proteins may be supplied as active agents to cells by a variety of means, including, for example, the introduction of nucleic acids, such as DNA or RNA, which encode the protein and may be accordingly transcribed and/or translated within the host cell, the addition of exogenous protein, and other suitable means of administration as are known to those skilled in the art. In whatever form in which supplied, the active agent can be used either alone or in combination with other active agents, using pharmaceutical compositions and formulations of the active agent which are appropriate to the method of administration. Pharmaceutically acceptable excipients, i.e., vehicles, adjuvants, carriers or diluents, are well-known to those who are skilled in the art, and are readily available. The choice of excipient will be determined in part by the particular compound, as well as by the particular method used to administer the compound. Accordingly, there is a wide variety of suitable formulations which can be prepared in the context of the present invention. However, pharmaceutically acceptable excipients not altering the neurotrophic activity of the recombinant protein are preferred.
7. Treatment with PEDF
A. Treatment of Tumors
PEDF is administered, alone or in conjunction with other clinical (such as surgical) procedures. PEDF is administered by intravitreal, subretinal, intravenous or intramuscular injection or injected or applied at sites of tumor growth. The PEDF may be administered alone or in conjunction with compounds, such as polylactic acid, which facilitate a slow, xe2x80x9ctime releasexe2x80x9d of the PEDF. Typically, about 0.01 to about 10 xcexcg of PEDF at a concentration of about 1 to about 100 xcexcg/ml in a physiologic saline solution or other buffered solution is administered per dose, and about 0 to about 10 doses are administered per day. When time-release compounds are included in the composition, they are used at a concentration of about 1 to about 100 xcexcg/ml. Treatment is continued until cessation of tumor growth and/or tumor regression is observed.
Treatment with PEDF is effective for retinal tumors such as retinoblastoma, other neuronal tumors such as neuroblastoma, or tumors of non-neuronal origin. The treatment results in a cessation or reduction in the rate of cell division and a concomitant reduction in the rate of tumor growth, which in turn results in tumor regression.
B. Neurotrophic Treatment of Ocular Disease
PEDF is administered, alone or in conjunction with other clinical (such as surgical) procedures. PEDF is administered by intravitreal or subretinal injection. The PEDF may be administered alone or in conjunction with compounds such as polylactic acid which facilitate a slow, xe2x80x9ctime releasexe2x80x9d of the PEDF. Typically, about 0.01 to about 10 xcexcg of PEDF at a concentration of about 1 to about 100 xcexcg/ml in a physiologic saline solution or other buffered solution is administered per dose, and about 0 to about 10 doses are administered per day. When time-release compounds are included in the composition, they are used at a concentration of about 1 to about 100 xcexcg/ml. Treatment is continued until the progress of the pathology is halted and/or reversed.
Treatment with PEDF is directed at diseases of the neural retina, retinal pigmented epithelium, and other ocular tissue. Treatment results in enhanced survival and well-being of the photoreceptors and other ocular cells, prolonging their functional life span and delaying the onset of impaired vision and ultimate blindness.
C. Neurotrophic Treatment of Neuronal Cell Pathology
PEDF is administered, alone or in conjunction with other clinical (such as surgical) procedures. PEDF is administered by intravenous or intramuscular injection or application or injection at the site of neuronal cell pathology. The PEDF may be administered alone or in conjunction with compounds such as polylactic acid which facilitate a slow, xe2x80x9ctime releasexe2x80x9d of the PEDF. Typically, about 0.01 to about 10 xcexcg of PEDF at a concentration of about 1 to about 100 xcexcg/ml in a physiologic saline solution or other buffered solution is administered per dose, and about 0 to about 10 doses are administered per day. When time-release compounds are included in the composition, they are used at a concentration of about 1 to about 100 xcexcg/ml. Treatment is continued until nerve regeneration is completed.
Treatment with PEDF is directed at injuries to nerves and pathologies of cells. Treatment results in enhanced survival of nerve cells and promotion of neurite outgrowth and nerve regeneration.
D. Treatment of Conditions Related to Serine Proteinases
PEDF is a member of the serine proteinase inhibitor family of proteins. As such, it is effective in the treatment of conditions caused by serine proteinases or where a serine proteinase inhibitor would be advantageous. Serine proteinases include, but are not limited to, chymotrypsin, trypsin, subtilisin, elastin, thrombin, and plasmin. Therefore, PEDF is useful as: an anti-coagulant, an anti-thrombotic, an anti-microbial, an anti-fungal, an anti-parasitic, and a contraceptive; in cosmetic preparations as a proteinase inhibitor; as a weight-gain promoter; in treatments for elastosis, vascular disorders involving fibrinoid formation, coagulation disorders, arteriosclerosis, ischemia, arthroses diabetes, emphysema, arthritis, septic shock, lung diseases, excessive complement activation, ulcers, ulcerative colitis, pancreatitis, psoriasis, fibrinolytic disease, arthropathy, bone resorption, hypertension, congestive heart failure, cirrhosis, or allergy caused by proteases, as a dermatological agent for skin discoloration and age-related wrinkling.
For use as an anti-coagulant, an anti-thrombotic, an anti-microbial, an anti-parasitic, or a contraceptive, or in treatment of elastosis, vascular disorders involving fibrinoid formation, coagulation disorders, arteriosclerosis, ischemia, arthroses diabetes, emphysema, arthritis, septic shock, lung diseases, excessive complement activation, pancreatitis, psoriasis, fibrinolytic disease, arthropathy, bone resorption, hypertension, congestive heart failure, cirrhosis, or allergy caused by proteases, PEDF is administered by intravenous or intramuscular injection or site-directed injection or application. The PEDF may be administered alone or in conjunction with compounds such as polylactic acid which facilitate a slow xe2x80x9ctime releasexe2x80x9d of the PEDF. Typically, about 0.01 to about 10 xcexcg of PEDF at a concentration of about 1 to about 100 xcexcg/ml in a physiologic saline solution or other buffered solution are administered per dose, and about 0 to about 10 doses are administered per day. When time-release compounds are included in the composition, they are used at a concentration of about 1 to about 100 xcexcg/ml. Treatment is continued until progress of the pathology is halted and/or reversed.
Treatment with PEDF is directed at injuries to nerves. Treatment results in promotion of neurite outgrowth and nerve regeneration.
For use in cosmetic preparations as a proteinase inhibitor, arthritis or elastosis PEDF is added to the preparations to a concentration of about 0.01 to about 100 xcexcg/ml.
For use in treatment as a weight-gain promoter, ulcers, ulcerative colitis, or pancreatitis, PEDF may be administered orally at a concentration of about 0.01 to about 10 xcexcg per kg of body weight per day.
For use as a treatment for conditions such as psoriasis, PEDF may be administered topically at a concentration of about 0.01 to about 100 xcexcg/ml, formulated in a suitable carrier.